Isolated DNA molecule encoding human TSC403

ABSTRACT

This invention provides the TSC403 gene having a nucleotide sequence coding for the amino acid sequence of SEQ ID NO:1, which is a novel gene of great utility particularly in the field of research, diagnosis, therapy, etc. for cancer of the lung, among other diseases. 
     In addition, this invention provides the human ING1L gene comprising a nucleotide sequence coding for the amino acid sequence of SEQ ID NO:4, which is a novel human gene useful for regulating the cell cycle, inhibiting or activating cell proliferation, studies on metabolic aging or apoptosis of cells, pathological exploration, diagnosis and therapy of cancer and other diseases, and screening for the development of new drugs.

TECHNICAL FIELD

The invention relates to a gene of use as an index in the prophylaxis,diagnosis and therapy of human diseases and more particularly to a novellung-specific human gene which is homologous to human 1 amp-1 and -2[lysosomal membrane glycoprotein; Saito, O. et al., J. Biol. Chem., 267,5700-5711 (1992); Sawada, R. et al., J. Biol. Chem., 268, 12675-12681(1993); Sawada, R. et al., J. Biol. Chem., 269, 1425-1431 (1994)] andsuspected to act as an oncogene.

The invention further relates to a novel human gene which is analogousto the rat, mouse, yeast, nematode, known human and other genes and,through the cDNA analysis, chromosome mapping and functional analysis ofits cDNA, can be utilized in gene diagnosis and for the development ofnew therapeutic drugs.

In addition, the invention relates to novel proteins encoded by saidgenes and to specific antibodies thereto.

BACKGROUND ART

The genetic information in organisms is accumulated as arrays (DNA) offour kinds of bases, viz. A, C, G and T, in the cell nucleus, and thisgenetic information is conserved for maintenance of lineage andontogenesis. In a human being, the number of such bases is said to beapproximately three-billion (3×10⁹) and it is estimated that thispopulation includes 50-100 thousand genes. The genetic information isinvolved in the maintenance of vital phenomena through the creation ofregulatory proteins, structural proteins, enzymes, etc. along the flowof transcription of mRNA from genes (DNA) and ensuing translation intoproteins.

It is generally acknowledged that any abnormality of the above flow froma gene to its translation product protein leads to an error of the lifemaintenance system inclusive of the proliferation and differentiation ofcells, and can becauses of various diseases. The results of geneanalyses made to this day suggest that the genes of various receptors,such as the insulin, LDL and other receptors, and those of metabolicenzymes associated with the growth and differentiation of cells, such asprotease, ATPase, superoxide dismutase, etc., are considered to beuseful tools for the development of pharmaceuticals.

However, the analysis of human genes and the study of their functionsand relationships to various diseases are still in the inchoate stageand much remains to be known. Therefore, analysis of new genes,analytical explorations into the functions and relationships to diseasesof such genes, and studies for the establishment of gene diagnosticsexploiting the genes so analyzed, and pharmaceutical application studieson such genes are subjects of immense interest to this industry.

Meanwhile, carcinoma of the pancreas is one of the malignant tumors ofthe digestive system with the poorest prognosis, ranking fourth andfifth on the list of causes for cancer-related death in Japan andWestern counties, respectively (Poston, J. G., et al., Gut., 32, 800-812(1991)). The most important goal in cancer research is to identifychanges in the genes in the early phase of oncogenesis. Identificationof such changes should lead to the development of genetic tools forearly diagnosis and novel therapeutic modalities for effective treatmentof this lethal disease.

Elucidation of the physiological roles of such genes and the resultinginformation are important to the explication of the mechanisms ofgenesis and onset of neoplastic diseases, and have been demanded notonly in the field of fundamental scientific research but also from thestandpoint of characterization and treatment of malignant tumors in thepharmaceutical field.

DISCLOSURE OF INVENTION

Thus, assuming that a novel human gene be provided, its expressionlevels in various cells as well as its structure and functions could beelucidated and through analysis of expression products of the gene, theclarification of pathology, diagnosis and therapy of the diseasesassociated with the gene, such as hereditary diseases and cancers, wouldbecome feasible. The object of the invention is to provide such novelhuman genes.

With the above object in mind, the inventors did intensive research asdescribed below. Thus, to begin with, the inventors synthesized cDNAsfrom the mRNAs extracted from various human tissues such as human fetalbrain, adult blood vessel and placenta, cloned them into vectors toconstruct libraries, cultured Escherichia coli cells transformed witheach library on agar medium picked up transformant colonies at randomand transferred them to microtiter plates to prepare and register E.coli clones containing various human genes. Then, each of these cloneswas cultured, the DNA extracted and purified, and using the cDNA thusobtained as a template, an amplification reaction with chain terminationspecific to said 4 bases is carried out by the deoxy terminator method,and using an automatic DNA sequencer, the sequence of about 400nucleotides from the 5′ end of the human gene in each registered clonewas determined. Based on the thus-obtained nucleotide sequenceinformation on human genes, novel family genes similar to the knownbacterial, yeast, nematoid, murine, human and other animal and plantgenes were explored. The above technology for cDNA analysis is describedin detail in the report of Fujiwara et al. [Fujiwara, Tsutomu, SaiboKogaku (Cell Engineering), 14, 645-654 (1995)].

As a result, among the cDNA clones picked up arbitrarily from the humanfetal brain cDNA library, the inventors found a clone harboring a novelgene which codes for an amino acid sequence having high homology top33^(ING1) which is considered to be a cancer-suppressive protein[GenBank A. C. No. AF001954, Garkavetsev, et al., Nature, Genet., 14,415-420 (1996); Garkavetsev, et al., Mol. Cell. Biol., 17, 2014-2019(1997); rewrote-GenBank A. C. No. AF0440767]. This invention has beendeveloped on the basis of the above finding.

Furthermore, for the purpose of providing said information demanded bythe industry, in particular a gene coding for a novel protein havinghomology to lamp-1 gene and lamp-2 gene, the inventors made an intensiveexploration into the genes derived from various human tissues andsucceeded in isolating and characterizing a novel lung-specific genematching for the above purpose. This invention has been developed on thebasis of the above finding.

Thus, in the first place, the invention provides a gene containing anucleotide sequence coding for the amino acid sequence of SEQ ID NO:1(hereinafter referred to TSC403 gene), in particular said gene which isa human gene.

In addition, the invention provides a novel protein encoded by saidTSC403 gene (hereinafter referred to as TSC403 protein) and an antibodyhaving a binding affinity for said protein.

Further, the invention provides a TSC403 gene which is any one of thefollowing polynucleotides (a), (b) and (c), particularly said gene whichis a human gene.

(a) a polynucleotide containing the nucleotide sequence of SEQ ID NO:2or a complementary chain there; to

(b) a polynucleotide which hybridizes to a DNA having the nucleotidesequence of SEQ ID NO:2 under stringent conditions; and

(c) a polynucleotide having at least 95% homology to a polynucleotidecoding for a polypeptide containing the amino acid sequence of SEQ IDNO:1

The invention further provides a TSC403 gene having the nucleotidesequence of SEQ ID NO:3.

The invention further provides an oligonucleotide having a sequenceconsisting of at least 15 consecutive nucleotides in the nucleotidesequence of SEQ ID NO:2 and a DNA fragment for use as a specific probeor primer for detecting genes having said oligonucleotide sequence.

Furthermore, in accordance with the invention, there is provided a humangene (hereinafter referred to as human ING1L gene) containing anucleotide sequence coding for the amino acid sequence of SEQ ID NO:5.

This invention further provides a protein (hereinafter referred to ashuman ING1L protein) which is encoded by said human ING1L gene and anantibody binding said protein.

Further provided in accordance with this invention is a human ING1L genecomprising any one of the following polynucleotides (a), (b) and (c).

(a) a polynucleotide containing the nucleotide sequence of SEQ ID NO:6;

(b) a polynucleotide containing a nucleotide sequence which hybridizesto a DNA having the nucleotide sequence of SEQ ID NO:6 under stringentconditions; and

(c) a polynucleotide having at least 95% homology to a polynucleotidecoding for a polypeptide containing the amino acid sequence of SEQ IDNO:5.

Further provided in accordance with this invention is a human ING1L genehaving the nucleotide sequence of SEQ ID NO:7.

In addition, according to the invention, there are provided anoligonucleotide having a sequence consisting of at least 15 consecutivenucleotides in the nucleotide sequence of SEQ ID NO:6 and a DNA fragmentfor use as a specific probe or primer for detecting genes having saidoligonucleotide sequence.

Representation of amino acids, peptides, nucleotide sequences,nucleotides, etc. by abbreviations in this specification is inconformity with the rules recommended by IUPAC-IUB [IUPAC-IUBCommunication on Biological Nomenclature, Eur. J. Biochem., 138, 9(1984)], “Guideline for Preparation of a Specification or EquivalentReferring to a Nucleotide Sequence and/or an Amino Acid Sequence”(edited by the Patent Office of Japan) and the conventions relating tothe use of codes or symbols in the art.

The TSC403 gene according to the invention is now described in detail.

As a specific example of the TSC403 gene according to the invention,there can be mentioned the gene deduced from the DNA sequence of a PCRproduct named “TSC403” as described in the Example which appearshereinafter. Its nucleotide sequence is presented in SEQ ID NO:3.

This gene is a human cDNA coding for a novel lung-specific proteinhaving a sequence of 416 amino acid residues as shown in SEQ ID NO:1(hereinafter referred to as “TSC403 protein”) and this cDNA has a fulllength of 3198 nucleotides.

The TSC403 protein of the invention occurs as an expression product ofthe gene of the invention. A homology search using FASTA Program[Person, W. R., et al., Proc. Natl. Acad. Sci., USA, 85, 2444-2448(1988)] against the GenBank/EMBL database revealed that this gene ishomologous to human lamp-1 gene and lamp-2 gene (cf. the literaturecited above).

In this connection, it is known that said human lamp genes are expressedat high levels in a highly metastatic colorectal cancer cell line andbind to E-selectin on the vascular endothelial cell. It is, therefore,suspected that these genes are associated with the malignancy of cancers(the literature cited above).

The TSC403 gene according to the invention is also a cancer-relatedgene, which is expected to find application as a cancer marker.

Furthermore, the chromosomal locus of this gene of the invention is 3q27where chromosomal aberration is detected in various cancers. This fact,even alone, strongly suggests the relation this gene of the inventionhas to various neoplastic diseases.

Furthermore, the TSC403 gene according to the invention was found toshow high expression in various cancer specimens, suggesting its valueas a marker for predicting oncogenesis and malignancy.

Thus, the TSC403 gene or a gene product thereof in accordance with theinvention provides the information or means of immense importance to theelucidation. understanding, diagnosis, prophylaxis and therapy ofvarious neoplastic diseases such as colorectal cancer, cancer of theuterus, cancer of the ovary, cancer of the lung, and cancer of thepancreas, among others. Furthermore, this gene of the invention can beused with advantage in the development of new drugs which would induceexpression of the gene for use in the treatment of said neoplasticdiseases.

In addition, detection of expression of the gene of the invention orexpression of its product in an individual or a given tissue as well asdetection of amutation (deletion or point mutation) or expressionabnormality of said gene can be exploited to advantage in theexplication and diagnosis of said various neoplastic diseases.

The human ING1L gene of the invention is now described in detail.

As a specific example of the human ING1L gene according to theinvention, there can be mentioned the gene deduced from the DNA sequenceharbored by the clone named “GEN-146F11” and described in the Examplewhich appears hereinafter. The nucleotide sequence of this gene ispresented in the SEQUENCE LISTING. Thus, the gene harbored by this clonehas a 840 -nucleotide open reading frame (deduced amino acid translatedregion; the sequence is shown in SEQ ID NO:6) which codes for thesequence of 280 amino acid residues as shown in SEQ ID NO:5 in theSEQUENCE LISTING, and the full-length nucleotide sequence of the cDNAclone consists of 1078 nucleotides as shown in SEQ ID NO:7.

In the above sequence of SEQ ID NO:7, the initiation codon is located inthe position 92-94 and the termination codon in the position 932 -934.The polyadenylation signal-like sequence (ATTAAA) is located in theposition 1058-1063.

As mentioned above, the human ING1L gene of the invention has highhomology to p33^(ING1) and can be utilized in the analysis of humangenes based on its genetic information and studies on the relationshipsof various functions of the genes so analyzed to various diseases andfurther exploited in the gene diagnosis and gene therapy of thegene-related diseases and application studies on the genes in thepharmaceutical field. Thus, the functions of the protein (gene product)encoded by the human ING1L gene of the invention can be predicted fromthose of the known homologous genes, and as the result of provision ofthe gene of the invention, it is now possible to construct a recombinantprotein by cloning the candidate gene in an expression vector andinvestigate its enzymatic activity, binding activity and otherfunctions. Particularly, since the gene of the invention is suspected tofunction as an oncogene, this function can be utilized with advantage inthe development of pharmaceuticals such as anticancer drugs.

The protein (hereinafter referred to as “human ING1L protein) encoded bythe human ING1L gene of the invention has a Zn finger motif-likesequence in its C-terminal region and this region in particular isconsidered to have high homology to said p33^(ING1).

In this connection, it has been reported that said p33^(ING1) isinactivated in several cancer-derived cell lines including a mammarycancer cell line (the literature cited above). Moreover, it has recentlybeen demonstrated that said p33^(ING1) is negatively regulating cellproliferation through p53 which is known to be a cancer-suppressive geneproduct [Garkavetsev, etal., Nature, 391, 295-298 (1998)]. Furthermore,in various human neoplastic tissues, the expression level of human ING1Lgene is specifically elevated. From these findings, it is suspected thatthe human ING1L protein is positively modulating cell proliferationthrough its interaction with p53.

Furthermore, in Northern blot analysis, expression of the human ING1Lgene of the invention was found in all the 16 human adult organ-derivedtissues tested and enhancement of its expression was noted in severalneoplastic tissues including colorectal cancer, cancer of the esophagus,cancer of the uterine tube, and stomach cancer as compared with thenormal tissues. These findings suggest that the gene of the inventioncan be used for the diagnosis of neoplastic and other diseasesassociated with it by checking for the expression thereof in varioustissues and, as a corollary, finds application in the screening forantimitotic compounds or anticancer compounds.

The gene of the invention specifically includes polynucleotidescontaining the nucleotide sequences of SEQ ID NOS:2 and 6 which code forthe amino acid sequences of SEQ ID NOS:1 and 5, respectively,polynucleotides which hybridize to DNAs containing the nucleotidesequences of SEQ ID NOS:2 and 6 under stringent conditions, andpolynucleotides having at least 95% homology to polynucleotide encodingthe amino acid sequences of SEQ ID NOS: 1 and 5.

Therefore, the gene of the invention includes those genes which encodeamino acid sequences corresponding to certain modifications of theabove-defined amino acid sequences and those genes which have a defineddegree of homology to the above-defined nucleotide sequences.

Thus, the gene of the invention includes, among others, genes containingnucleotide sequences coding for the amino acid sequences resulting fromthe deletion, substitution or addition of one or a plurality of aminoacids from, in or to the amino acid sequence of SEQ ID NO:1 or 5 (i.e.modified amino acid sequences). The gene having a nucleotide sequencecoding for such a modified amino acid sequence need only be such that byutilizing it, the gene of the invention coding for the unmodified aminoacid sequence can be detected.

Incidentally, while such modifications (mutation etc.) of amino acidsequences may be spontaneous, e.g. mutations and post-translationalmodifications, the modifications can be made artificially as well byutilizing a gene of the natural origin (for example, a specific gene ofthe invention).

The means for making such artificial modifications includes geneticengineering techniques such as site-specific (-directed) mutagenesis[Methods in Enzymology, 154:350,367-382(1987); ditto 100:468(1983);Nucleic Acids Res., 12: 9441 (1984); Zoku Seikagaku Jikken Koza 1“Idenshi Kenkyuho II” [Experimental Biochemistry Series 1 “Methods forGene Research II” (edited by Japanese Biochemical Society), p105 (1986)], etc. and chemical synthetic techniques such as the phosphotriestermethod and the phosphoamidate method [J. Am. Chem. Soc., 89: 4801(1967); ditto 91: 3350 (1968); Science, 150: 178 (1968); TetrahedronLett., 22: 1859 (1981); ditto 24: 245 (1983)] as well as a suitablecombination of such techniques.

As one example of the gene according to the invention, the genecomprising a polynucleotide having the nucleotide sequence of SEQ IDNO:2 or 6 or a complementary sequence thereto can be mentioned. Thisnucleotide sequence represents an example of combination of codons foreach amino acid residue of the above amino acid sequence (SEQ ID NO:1 or5). Of course, the gene of the invention is not limited to the abovecombination but the gene having a nucleotide sequence designed by.selecting an arbitrary combination of codons for each of said amino acidresidues can be employed. Selection of said codons can be made in theroutine manner. In this selection, the codon frequency of the host to beused may be taken into consideration [Nucleic Acids Res., 9: 43 (1981)].

Furthermore, while the gene of the invention is shown as the nucleotidesequence of a single-stranded DNA as, for example, shown in SEQ ID NO:3or 7 the invention of course encompasses a polynucleotide comprising anucleotide sequence complementary to such a nucleotide sequence and acomponent containing both of them as well and, moreover, is not limitedto a DNA such as cDNA.

Furthermore, as mentioned above, the gene of the invention is notlimited to one comprising a polynucleotide having the nucleotidesequence of SEQ ID NO:2 or 6 or a complementary sequence thereto butincludes one comprising a nucleotide sequence having a given degree ofhomology to such a nucleotide sequence. More particularly, there isincluded the gene comprising a polynucleotide having at least 95%homology to a polynucleotide coding for a polypeptide having the aminoacid sequence of SEQ ID NO:1 or 5.

Moreover, the gene of said nucleotide sequence having a defined homologyincludes one that hybridizes to a DNA having the nucleotide sequence ofSEQ ID NO:2 or 6 under stringent conditions such as those describedbelow and does not lose the DNA even when the hybrid is washed undergiven conditions.

As an example, there can be mentioned a gene having a nucleotidesequence which, when hybridized to a DNA having the nucleotide sequenceof SEQ ID NO:2 or 6 in 6×SSC at 65° C. overnight or in 4×SSCsupplemented with 50% formaldehyde at 37° C. overnight and, then, washedin 2×SSC at 65° C. for 30 minutes, will not be disengaged from the DNA.Here, SSC stands for standard saline-citrate buffer (standard salinecitrate; 1×SSC=0.15 M NaCl, 0.015 M sodium citrate). A preferred exampleof said gene is a gene having a nucleotide sequence which, even whenhybridized to a DNA having the nucleotide sequence of SEQ ID NO:2 or 6in 7% polyethylene glycol (PEG)/10% sodium dodecyl sulfate (SDS) at 65°C. overnight and washed in 0.1×SSC/0.1% SDS at 65° C. for 30 minutes,will not be disengaged from the DNA.

The gene of the invention can be easily produced and acquired by thestandard genetic engineering techniques [Molecular Cloning 2d Ed, ColdSpring Harbor Lab. Press (1989); Zoku Seikagaku Jikaen Koza “IdenshiKenkyuho I, II, III” [New Experimental Biochemistry Series “Methods forGene Research I, II, III” (edited by Japanese Biochemical Society),(1986), etc. ] based on the sequence information on the specificexamples shown in SEQ ID NO:3 or 7.

More particularly, the objective gene can be acquired by constructing acDNA library from a suitable source containing the gene of the inventionand selecting the desired clone from this cDNA library using a suitableprobe or antibody specific to the gene of the invention in the per seknown manner [Proc. Natl. Acad. Sci., USA., 78: 6613 (1981); Science,222: 778 (1983), etc.].

In the above procedure, the cDNA source includes to various cells ortissues in which the gene of the invention is expressed and culturedcells derived therefrom. Particularly in the case of the TSC403 gene ofthe invention, lung tissues can be mentioned by way of example.Isolation of the whole RNA from such a source, isolation andpurification of mRNA, synthesis of cDNA, and cloning thereof can all becarried out in the routine manner. cDNA libraries are also commerciallyavailable. In the practice of the invention, such commercial cDNAlibraries, for example those available from Clontech Lab. Inc., can alsobe employed.

The method of screening for the gene of the invention from a cDNAlibrary is not particularly restricted, either, but a conventionalmethod can be selectively employed. To be specific, selection of a cDNAclone by an immunoscreening technique using a specific antibody againstthe protein produced by the cDNA, the plaque hybridization or colonyhybridization technique using a probe having a selective bindingaffinity for the objective DNA sequence, or a combination thereof can bementioned by way of example.

As to the probe to be used in the above procedure, it is generallyuseful to use a DNA chemically synthesized according to the nucleotidesequence information on the gene of the invention. Of course, it is alsopossible to use the gene already obtained or a fragment thereof as saidprobe.

The nucleotide sequence which can be used as said probe includes apartial nucleotide sequence corresponding to SEQ ID NO:2 or 6 butconsisting of at least 15 consecutive nucleotides, preferably within therange of 20-30 nucleotides. Moreover, positive clones containing theabove respective sequences can also be utilized as said probe.

Said screening can be carried out by the procedure which uses, as thescreening probe, a set of sense and antisense primers based on thepartial amino acid sequence information about a natural extract isolatedand purified from a given cell line or tissue.

Furthermore, said screening can also be carried out by the proteininteraction cloning procedure using the TSC403protein or human ING1Lprotein in lieu of said specific antibody.

In the invention, the expression of mRNA in cells under differentconditions or between a plurality of cell groups can be studied bydirect comparison using the differential display method [Liang, P., etal., Science, 257, 967-971 (1992)].

In obtaining the gene of the invention, DNA/RNA amplification by PCR[Science, 230, 1350 (1985)] can also be used with advantage.Particularly in case where a full-length cDNA can hardly be obtainedfrom a library, the RACE [rapid amplification of cDNA ends] method[Jikken Igaku (Experimental Medicine), 12(6): 35 (1994)], in particularthe 5′-RACE method [Frohman, M. A., et al., Proc. Natl. Acad. Sci.,USA., 8: 8998 (1988)], can be used with advantage. The primers for usein such PCR methods can be judiciously established according to thesequence information on the gene of the invention and can be synthesizedby the conventional procedure.

Isolation and purification of the amplified DNA/RNA fragment can becarried out by the conventional techniques as mentioned hereinbefore,for example by gel electrophoresis.

The nucleotide sequence of the gene of the invention or any of variousDNA fragments thereof can be determined in the routine manner, forexample by the dideoxy method [Proc. Natl. Acad. Sci., USA., 74: 5463(1977)], the Maxam-Gilbert method [Methods in Enzymology, 65: 499(1980)] or, more expediently, by means of a commercial sequencing kit.

With the gene of the invention, the gene product can be produced easily,on a high production scale, and with good reproducibility by thestandard genetic engineering technology.

The invention further provides a vector (expression vector) harboringsaid TSC403 gene or human ING1L gene, host cells transformed by usingsaid vector, and a method of producing TSC403 protein or human ING1Lprotein which comprises growing said host cells.

Production of said TSC403 protein and human ING1L protein can be carriedout by the standard recombinant DNA technology [Science, 224: 1431(1984): Biochem. Biophys. Res. Comm., 130: 692 (1985): Proc. Natl. Acad.Sci., USA., 80: 5990 (1983), and the reference literature citedhereinabove].

As said host cells, whichever of prokaryotic cells and eucaryotic cellscan be employed. As the prokaryotic host, various procaryotes which arecommonly employed, such as Escherichia coli and Bacillus subtilis, canbe liberally employed. The preferred host cells are those derived fromEscherichia coli, particularly cells of E. coli K12.

The eucaryotic host cells include cells of vertebrate and yeasts, amongothers. Among the former cells, the monkey cell line COS [Cell, 23: 175(1981)], Chinese hamster ovarian cells and the dihydrofolatereductase-defective line thereof [Proc. Natl. Acad. Sci., USA., 77: 4216(1980)] can be mentioned as examples. As to the latter cells, cells ofyeasts belonging to the genus Saccharomyces can be mentioned as examplesbut these are not exclusive choices.

When prokaryotic cells are used as host cells, a vector which can bereplicated in the host cell is selected and, for expression of the gene,an expression plasmid provided with a promoter and the SD(Shine-Dalgarno) sequence upstream of the gene of the invention, as wellas an initiation codon (e.g. ATG) necessary to start protein synthesis,can be employed with advantage. As the vector mentioned above, it isusual to employ an E. coli-derived plasmid, such as pBR322, pBR325,pUC12, pUC13, etc., although these are not exclusive choices and variousknown other vectors may be utilized. As commercial vectors forexpression systems using E. coli, pGEX-4T (Amersham Pharmacia Biotech),pMAL-c2, pMAL-p2 (New England Biolabs), pET21, pET21/lacq (Invitrogen),pBAD/His (Invitrogen), among others, can be mentioned by way of example.

As the expression vector to be used when cells of a vertebral animal areemployed, usually a vector having a promoter region upstream of the geneto be expressed, RNA splice sites, polyadenylation site, transcriptionend sequence, etc. can be mentioned, and where necessary, the vectorfurther has a replication origin. As a specific example of the abovevector, pSV2dhfr containing an early promoter of SV40 [Mol. Cell. Biol.,1: 854 (1981)], for instance, can be mentioned. Aside from the above,various other known commercial vectors can be used. As commercialvectors which can be used in expression systems utilizing animal cells,there can be mentioned various vectors available for animal cell use,such as PEGFP-N, pEGFP-C (Clontech), pIND (Invitrogen), pcDNA3.1/His(Invitrogen), etc. and vectors available for insect cell use, such aspFastBacHT (Gibco BRL), pAcGHLT (PharMingen), pAc5/V5-His, pMT/V5-Hisand pMT/Bip/V5-His (all Invitrogen).

As a specific example of the expression vector which can be used whenyeast cells are used as the host cells, pAM82 having a promoter for theacid phosphatase gene [Proc. Natl. Acad. Sci., USA., 80: 1 (1983)] canbe mentioned. The commercial expresion vectors for yeast cell useinclude to pPICZ (Invitrogen) and pPICZ (Invitrogen).

The promoter is not particularly restricted, either. When a bacterialstrain of the genus Escherichia is used as the host, tryptophan (trp)promoter, lpp promoter, lac promoter, recA promoter, PL/PR promoter,etc. can be used with advantage. When the host is an organism of thegenus Bacillus, SP01 promoter, SP02 promoter, penP promoter, etc. arepreferred choices. The promoter which can be used with advantage when ayeast is used as the host includes pH05 promoter, PGK promoter, GAPpromoter and ADH promoter, among others. The preferred promoter in caseswhere animal cells are used as said host cells includes SV40 -derivedpromoter, retrovirus promoter, metallothionein promoter, heat shockpromoter, cytomegalovirus promoter and SR promoter, among others.

As expression vectors for the gene of the invention, the conventionalfusion protein expression vector can also be used with advantage. As anexample of the vector of this type, pGEX (Promega) for expression of afusion protein with glutathione-S-transferase (GST) can be mentioned.

The method of introducing said objective recombinant DNA (expressionvector) into the host cell (transformation method) is not particularlyrestricted, either, but various standardized methods can be utilized.Culture of the resultant transformant can also be performed in theroutine manner. By such culture, the objective protein encoded by thegene of the invention is expressed, produced, and accumulated in thetransformant cell or secreted extracellularly or on the cell membrane.

The medium for said culture can be judiciously selected from among theconventional media according to the type of host cells adopted, andculture can also be carried out under conditions suited forproliferation of the host cells.

The recombinant protein thus produced can be optionally isolated andpurified by various isolation procedures utilizing its physical,chemical or other properties [Seikagaku (Biochemical) Data Book II,pp.1175-1259, 1st Ed., 1st Impression, Jun. 23, 1980, Tokyo KagakuDojin; Biochemistry, 25(25): 8274 (1986); Eur. J. Biochem.,163:313(1987); etc.]. The procedures mentioned above specificallyinclude the standard reconstitution treatment, treatment with a proteinprecipitating agent (salting out), centrifugation, osmotic shock method,sonic disruption, ultrafiltration, various kinds of chromatography, e.g.molecular sieves chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, affinity chromatography,high performance liquid chromatography (HPLC), etc., dialysis, and theircombinations. The particularly preferred procedure is affinitychromatography using a column conjugated with a specific antibodyagainst the TSC403 protein or human ING1L protein according to theinvention.

The invention further provides the novel TSC403 protein or human ING1Lprotein obtainable as above and the technology of producing thoseproteins. The protein according to the invention finds application inthe pharmaceutical field as mentioned hereinbefore.

Moreover, the protein of the invention can be used as an immunogen forconstruction of a specific antibody against said protein. The componentfor use here as the antigen may be the protein produced in a largeamount by any of said genetic engineering techniques or a fragmentthereof, and by using such an antigen, the objective antiserum(polyclonal antibody) and monoclonal antibody can be obtained.

The production technology for such antibodies is well known to thoseskilled in the art and the production of antibodies relevant to theinvention can also be made in accordance with such establishedtechnology (Zoku Seikagaku Koza “Men-eki Seikagaku Kenkyuho” (NewImmunobiochemistry Series, “Methods in Immunobiochemistry”), edited byJapanese Biochemical Society (1986), among others].

For example, the immune animal for use in harvesting the antiserum canbe liberally selected from among ordinary animals such as the rabbit,guinea pig, rat, mouse, chicken, goat and sheep, and immunization withsaid antigen and collection of blood can also be carried out in theroutine manner.

Preparation of said monoclonal antibody can also be carried out in theroutine manner, i.e. by constructing a fusion cell from the plasma cell(immune cell) of an animal immunized with said immunogen and aplasmocytoma cell, selecting a clone producing the objective antibody,and growing the clone. The immune animal is generally selected inconsideration of its compatibility with the plasmocytoma cell to be usedfor cell fusion and usually the mouse or the rat is used with advantage.Immunization can be carried out in the same manner as in the preparationof said antiserum, and optionally the usual adjuvant can be used incombination with the antigen.

The plasmocytoma cell to be used for said fusion is not particularlyrestricted but may be any of various myeloma cells such as p3(p3/x63-Ag8) [Nature, 256:495-497(1975)], p3-U1 [Current Topics inMicrobiology and Immunology, 81: 1-7 (1978)], NS-1 [Eur. J. Immunol., 6:511-519 (1976)], MPC-11 [Cell, 8: 405-415 (1976)], SP2/0 [Nature, 276:269-271 (1978)], etc., R210 [Nature, 277: 131-133 (1979)], etc. fromrats and cells derived therefrom.

The fusion between said immune cell and plasmocytoma cell can beeffected in the presence of a conventional fusion promoter, such aspolyethylene glycol (PEG), Sendai virus (HVJ) or the like, in accordancewith a known protocol. Isolation of the objective hybridoma can also becarried out by the known procedure [Meth. in Enzymol., 73: 3 (1981);said Zoku Experimental Biochemistry Series; etc. ].

The search for the objective antibody-producing cell line and thepreparation of a monoclonal antibody can also be carried out in theconventional manner. For example, the search for an antibody producingline can be made by various techniques which are generally used fordetection of antibodies, such as ELISA [Meth. in Enzymol., 70: 419-439(1980)], plaque method, spot method, agglutination reaction method,Ouchterlony method, radioimmunoassay, etc., using the protein of theinvention as an antigen.

Isolation of the antibody of the invention from the hybridoma obtainedas above can be carried out by the method which comprises growing thehybridoma in the routine manner and recovering the antibody as a culturesupernatant or the method which comprises administering the hybridoma toa compatible mammal to let it multiply in vivo and recovering theantibody in the form of an ascite fluid. The former method is suitablefor the preparation of a high-purity antibody, while the latter methodis suited for high production. The antibody produced in this manner canbe purified by the routine procedure such as salting out, gelfiltration, affinity chromatography o r the like.

The antibody thus obtained is characterized in that it is capable ofbinding the protein of the invention. This characteristic can beexploited for the purification of the protein of the invention and theassay and identification of the protein by an immunological technique.The invention further provides such a novel antibody.

Based on the sequence information on the gene of the invention, whichhas been generated by the invention, the expression of the gene of theinvention in the individual or in various tissues can be detected byutilizing a part or the whole of the nucleotide sequence of said gene.

In the invention, for the purpose of detecting the presence of a TSC403gene or human ING1L gene whose expression level is elevated in a cancertissue, one may prepare a biological sample, such as a blood or serumsample, optionally extract the DNA, and carry out an analysis to seewhether the sample contains a susceptible TSC403 gene or human ING1Lgene.

In accordance with the invention, for the purpose of detecting thepresence of a marker of malignancy in cells or tissues, progression ofmalignancy to a prodromal disturbance, or prognosis, a biological sampleof malignancy is prepared and analyzed for the presence of a TSC403 orhuman ING1L oncogene. By utilizing this technique, the presence of sucha marker of malignancy in cells or a tissue, progression of malignancyto a prodromal disturbance, or prognosis can be detected. Therefore, theinvention enables the diagnosis of a cancer, evaluation of the effect ofa cancer therapy or prediction of the prognosis of a cancer.

The above detection can be carried out as follows. For example, based onthe information on TSC403 gene or human ING1L gene as obtained by usinga sample from a tumor-bearing patient, a DNA fragment designed for usein the screening for TSC403 gene or human ING1L gene and/or theamplification of the gene is first prepared. The DNA fragment mentionedabove includes the following.

(1) The fragment having the nature of a probe for plaque hybridization,colony hybridization, Southern blotting, Northern blotting, etc.

(2) The fragment having the nature of a probe for preparation of theentire or partial DNA fragment of TSC403 gene or human ING1L gene asamplified by PCR, that is a polymerase chain reaction for amplifying anucleotide sequence with a polymerase.

For the construction of such DNA fragments, a primer having the samesequence as TSC403 gene or human ING1L gene is first prepared. Usingthis primer as a screening probe, it is reacted with a biological sample(nucleic acid sample) to confirm the presence of a gene having theTSC403 gene sequence or human ING1L gene sequence.

The above nucleic acid sample can be prepared by various methodsproviding for easy detection of the target sequence, such asdenaturation, restricted digestion, electrophoresis or dot blotting.

The method of said screening is preferably PCR from the standpoint ofsensitivity. This method is not particularly restricted inasmuch as itemploys a TSC403 gene fragment or a human ING1L gene fragment as theprimer and it may be any of the known protocols [Science, 230: 1350-1354(1985)] and all PCR versions that are newly developed or expected to beused in the future [Sakaki, Y. et al. (ed.), Jikken Egaku (ExperimentalMedicine), Supplement 8(9) (1990), Yodosha; Protein·Nucleic Acid·Enzyme; Special Supplement, Kyoritsu Publishing Co., 35(17) (1990)].

The DNA fragments for use as primers are chemically synthesizedoligo-DNAs. Those oligo-DNAs can be synthesized by using an automaticDNA synthesizer, e.g. Pharmacia LKB Gene Assembler Plus (Pharmacia). Thelength of the primer (sense primer or antisense primer) may for examplebe the equivalent of about 10-50 nucleotides, more preferably about15-30 nucleotides.

The probe for use in the above screening is usually a labeled probe butmay be an unlabeled probe. The screening may depend on specific bindingwith a directly or indirectly labeled ligand. The method of labeling aprobe or a ligand is known in the art and the relevant prior artincludes nick translation, random priming, and kinase treatment, amongothers. The substance which can be used as the label includesradioisotopes, biotin, fluorescent groups, chemiluminescent groups,enzymes and antibodies which can be taken up by way of such knownmethods.

The above-mentioned detection can be performed in the routine manner.For example, RNA amplification by RT-PCR [reverse transcribed-polymerasechain reaction; E. S. Kawasaki, et al., Amplification of RNA. In PCRProtocol, A Guide to Methods and Applications, Academic Press, Inc.,SanDiego, 21-27 (1991)], Northern blot analysis [Molecular Cloning, ColdSpring Harbor Lab. (1989)], determination on the cellular level, e.g. insitu RT-PCR [Nucl. Acids Res., 21:3159-3166(1993)] and in situhybridization, NASBA method [nucleic acid sequence-based amplification,Nature, 350: 91-92 (1991)], modifications of these techniques which areknown in the art, and various other methods can invariably be used withsuccess.

The method for assay according to the invention can be carried outexpediently by utilizing an assay reagent kit for detection of TSC403gene or human ING1L gene in samples. The invention further provides anassay kit for detection of TSC403 gene or human ING1L gene whichcontains said TSC403 gene fragment or human ING1L gene fragment.

It is important that this assay kit contain at least a DNA fragmenthybridizing to a part or the whole of the nucleotide sequence of SEQ IDNO:2 or 6 or a complementary nucleotide sequence thereto as an essentialcomponent. As the other components, the kit may contain a labeling agentand reagents necessary for PCR, such as Taq DNA polymerase,deoxynucleotide triphosphate, and primers, among others.

The labeling agent includes radioisotopes and chemical modifiers such asfluorescent substances. These may be used as pre-conjugated to the DNAfragment.

For convenience in practice of the assay, the assay kit of the inventionmay contain a suitable reaction diluent, a standard antibody, a buffer,a washing buffer, a reaction stopper solution and so forth.

The invention further provides a method for cancer diagnosis utilizingthe above assay method, a diagnostic reagent for use in said diagnosis,and a diagnostic kit.

By sequencing the TSC403 gene or human ING1L gene in a test sample asobtained by using the above assay method of the invention, eitherdirectly or indirectly by the conventional procedure, it is possible todiscover a novel TSC403 or human ING1L-related gene (mutant gene) whichis highly homologous to the wild type TSC403 gene or wild type humanING1L gene. Therefore, the invention further provides a method ofscreening for a TSC403-related gene or human ING1L-related gene in atest sample which comprises performing said assay and sequencing theTSC403 gene or human ING1L gene in the sample.

Moreover, by utilizing the protein encoded by the TSC403 gene or humanING1L gene of SEQ ID NO:1 or 5, a protein having an amino acid sequenceresulting from the deletion, substitution or addition of one or aplurality of amino acids from, in or to said sequence of SEQ ID NO:1 or5, or an antibody against such a fragment (polyclonal antibody ormonoclonal antibody; hereinafter referred to as “TSC403 antibody” or“human ING1L antibody”), said wild type TSC403 gene, wild type humanING1L gene, mutant TSC403 gene and mutant human ING1L gene can besuccessfully assayed.

The invention further provides a method for assay of such a wild typeTSC403 gene, a wild type human ING1L gene, a mutant TSC403 gene or amutant human ING1L gene.

According to this assay methodology, the severity of disturbance in aneoplastic state or the malignancy of a neoplasm can be detected from achange in the wild type TSC403 gene or wild type human ING1L gene. Thechange mentioned above can be determined or detected by the sequencingof the TSC403 gene or human ING1L gene by any of the above-mentionedconventional sequencing techniques, and more preferably by the assaymethod using said TSC403 antibody or human ING1L antibody. In thismanner, the presence of an anomaly (mutation) in the TSC403 protein orhuman ING1L protein in a test sample or the presence or absence of theTSC403 protein or human ING1L protein can be detected.

In the assay procedure of the invention which utilizes an anti-TSC403antibody or anti-human ING1L antibody, the antibody can be used toimmunoprecipitate the TSC403 protein or human ING1L protein from asolution containing a biological material obtained from a human subject,such as blood or serum, or caused to react with TSC403 protein or humanING1L protein on the polyacrylamide gel Western blot or on theimmunoblot.

Furthermore, by utilizing the anti-TSC403 antibody or anti-human ING1Lantibody in an immunohistochemical assay procedure, the TSC403 proteinor human ING1L protein in a paraffin section or a frozen tissue specimencan be detected. The production technology and purification procedurewhich can be used for said anti-TSC403 antibody or anti-human ING1Lantibody are well known in the art. Such known techniques can beutilized for the production and purification of said antibody.

The more preferred protocol relevant to the detection of a wild typeTSC403 or human ING1L, or a mutant thereof, includes a sandwich assayusing amonoclonal antibody and/or a polyclonal antibody. Among otherpreferred detection techniques are enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA) andimmunoenzymometric assay (IEMA).

The invention further provides a TSC403 protein receptor or human ING1Lprotein receptor existing in a cell membrane fraction or a cell surfaceand having binding activity for TSC403 protein or human ING1L protein.This TSC403 protein receptor or human ING1L protein receptor can beproduced and obtained, for example by adding a labeled TSC403 protein orhuman ING1L protein to a cell membrane fraction containing the receptoror a biological sample containing the same, extracting, isolating andpurifying the resulting receptor-protein conjugate (TSC403protein-binding reaction product or human ING1L protein-binding reactionproduct) and identifying the amino acid sequence of the isolatedproduct. The preparation and sequencing of the TSC403 protein or humanING1L protein receptor can be easily made by those skilled in the artaccording to the established procedures

The TSC403 protein receptor or human ING1L protein receptor according tothe invention, or fragments thereof, can be applied to the screening forvarious drugs. By such technology, compounds capable of reacting withsaid receptor (low molecular compounds, high molecular compounds,proteins, protein fragments, antigens, antibodies, etc.) can be screenedout. The receptor, or a fragment thereof, which is to be used in suchscreening can be put to use as immobilized on a suitable solid matrix orin the form of a free substance in a solution transported to the cellsurface.

An example of the above pharmacoscreening is the screening in whichprocaryotic or eucaryotic host cells transformed stably with arecombinant protein expressing the TSC403 protein or human ING1Lprotein, or a fragment thereof, are used in, preferably, a competitivebinding assay. As an alternative, said host cells, whether in the freeform or as immobilized, are used in the standard binding assay. Moreparticularly, the above pharmacoscreening may comprise reacting theTSC403 protein receptor or human ING1L protein receptor, or a fragmentthereof, with the TSC403 protein or human ING1L protein, or a fragmentthereof, in the presence of a candidate drug, to cause formation of acomplex and detecting the degree of inhibition of the complex formationby the above candidate drug.

Thus, in accordance with the invention, there can be provided a methodfor pharmacoscreening which comprises contacting a candidate drug withthe TSC403 protein receptor or human ING1L protein receptor, or afragment thereof and, then, detecting the presence of the resultingcomplex or the presence of a complex of the TSC403 protein receptor orhuman ING1L protein receptor, or a fragment thereof, and the ligand by aper se known technique.

Furthermore, by assaying TSC403 protein receptor activity or human ING1Lprotein receptor activity, it is possible to evaluate whether acandidate drug is capable of antagonizing the TSC403 protein receptor orhuman ING1L protein receptor and accordingly inhibiting TSC403 proteinactivity or human ING1L protein activity, for example mitosis-promotingactivity.

In such a competitive binding assay, the TSC403 protein receptor orhuman ING1L protein receptor, or a fragment thereof, is labeled. Whenthe free TSC403 protein receptor, human ING1L protein receptor orfragment thereof is separated from the corresponding complex and thelabeling amount of the free (non-complex-forming) substance is measured,the measured value serves as a yardstick of the binding of the candidatedrug to the TSC403 protein receptor or human ING1L protein receptor.Furthermore, said measured value serves also as a measure of inhibitionof the binding of the TSC403 protein receptor or human ING1L proteinreceptor to the TSC403protein or human ING1L protein. By analyzing asmall peptide (pseudopeptide) of TSC403 protein or human ING1L proteinin this manner, the candidate drug can be assayed as a substance havingTSC403 protein receptor antagonizing activity or human ING1L proteinreceptor antagonizing activity.

Another protocol for pharmacoscreening in accordance with the inventionis that of screening for a compound having an adequate binding affinityfor the TSC403 protein receptor or human ING1L protein receptor.Briefly, this procedure comprises synthesizing a large number ofdifferent test peptide compounds on a solid support such as the surfaceof a plastic pin or other material, reacting the test compounds with theTSC403 protein receptor or human ING1L protein receptor and, afterwashing, detecting the binding reaction products by a known method [e.g.PCT patent publication No. WO 84-03564].

The purified TSC403 protein receptor or human ING1L protein receptor canbe directly coated on the plate to be used in said pharmacoscreeningprocedure. Moreover, the antibody may be captured with anon-neutralizing antibody against the polypeptide and the TSC403 proteinreceptor or human ING1L protein receptor be immobilized on a solidphase.

The invention is further directed to the use of a competitivepharmacoscreening assay. For the binding to the TSC403 protein receptoror human ING1L protein receptor, or a fragment thereof, a neutralizingantibody capable of specific binding to the TSC403 protein receptor orhuman ING1L protein receptor is caused to compete with the candidatecompound. By such a competitive reaction with the neutralizing antibody,the presence of any peptide having one or more antigenic determinants ofthe TSC403 protein receptor or human ING1L protein receptor can bedetected.

Furthermore, in connection with pharmacoscreening, a still anothermethod comprises the use of a host eucaryotic cell line or cellscontaining a nonfunctional TSC403 gene or nonfunctional human ING1Lgene. The host cell line or cells are caused to multiply in the presenceof a candidate drug for a predetermined time and the velocity of growthof the host cells is determined to see whether, for example, thecandidate drug is capable of inhibiting growth of the cells. The meansfor measuring said velocity of growth includes a method of determiningthe biological activity of the TSC403 protein receptor or human ING1Lprotein receptor.

Furthermore, in accordance with the invention, for the development of amore active or stable derivative of TSC403 protein or human ING1Lprotein or of a drug which will enhance or interfere with the functionof TSC403 protein or human ING1L protein in vivo, it is possible toconstruct bioactive proteins, or their structural analogs, with whichsaid protein would interact, such as a TSC403 protein or human ING1Lprotein receptor agonist, a TSC403 protein or human ING1L proteinreceptor antagonist, or a TSC403 protein or human ING1L proteininhibitor, for instance. The structural analogs mentioned above can becharacterized by analysis of the three-dimensional structure of acomplex between TSC403 protein or human ING1L protein and a third-partyprotein by X-ray crystallography or computer modeling or a combinationof such techniques. The structural information on such structuralanalogs can also be obtained by protein modeling based on the structuresof homologous proteins.

As a method for providing said more active or stable TSC403 proteinderivative or human ING1L protein derivative, there can be mentioned analanine scan technique. This technique comprises substituting an alanineresidue for each of certain amino acid residues of said protein anddetermining the effect of substitution on the activity of the resultingprotein. In other words, this technique is such that by saidsubstitution for amino acid residues of the protein and analysis, thedomain of significance to the activity or stability of the protein isdetermined. This technique enables design of a more active or stableTSC403 protein derivative or human ING1L protein derivative.

Furthermore, it is now possible to isolate a target-specific antibodyselected by a functional assay and analyze its crystal structure. As arule, the pharmacore providing a basis for subsequent drug design can beobtained by this approach. Through the generation of an anti-idiotypeantibody for a functional pharmacoactive antibody, a peptide can beidentified or isolated from a chemically or biologically constructedpeptide bank. Hence, the selected peptide is also expected to serve as apharmacore.

Thus, it is now possible to design and develop drugs having TSC403protein or human ING1L protein inhibitor, agonist or antagonist activityfor TSC403 protein or human ING1L protein having improved activity,stability and other characteristics.

It is also possible to prepare a sufficient amount of TSC403 protein orhuman ING1L protein by using a cloned TSC403 gene or cloned human ING1Lgene and carry out X-ray crystallographic and other analyticalinvestigations. Furthermore, as the result of provision of the TSC403protein or human ING1L protein of SEQ ID NO:1 or 5 according to theinvention, it is now possible to provide a computer modeling program ortechnique as a substitute for X-ray crystallography or as an adjunctthereto.

The invention enables construction of a TSC403 gene-bearing knockoutmouse (mutant mouse) or human ING1L gene-bearing knockout mouse (mutantmouse). By this approach, it can be ascertained which region of thenucleotide sequence of the TSC403 gene or human ING1L gene influencessaid divergent activities of TSC403 protein or human ING1L protein invivo, that is to say what functions TSC403 gene products, human ING1Lgene products, modified-TSC403 gene products or modified-human ING1Lgene products would have in vivo.

This is a technique for modifying the genetic information of an organismintentionally by utilizing a homologous recombination of genes, and aprotocol using mouse embryonic stem cells (ES cells) is known[Capeccchi. M. R., Science, 244, 1288-1292 (1989)].

The above construction of mutant mice belongs to the expertise of thosekilled in the art and the wild type TSC403 gene, wild type human ING1Lgene, mutant TSC403 gene or mutant human ING1L gene according to theinvention can be subjected to this modification [Tetsuo Noda (ed.):Jikken Igaku (Experimental Medicine), Supplement, 14(20) (1996),Yodosha] to thereby construct said respective mutant mice in anexpedient manner. By utilizing this technique, it is possible to designand develop drugs having TSC403 protein or human ING1L proteininhibitor, agonist or antagonist activity for the protein havingimproved TSC403 protein activity or stability or improved human ING1Lprotein activity or stability.

Thus, the invention further provides a specific primer for detection ofthe TSC403 gene or human ING1L gene of the invention and/or a DNAfragment for use as the specific primer, a method for cancer diagnosiswhich utilizes them, and a diagnostic kit therefor.

For example, the TSC403 gene probe according to the invention can beproduced and acquired by the standard PCR technique using two kinds ofprimers (sense primer and antisense primer) which are specific to theTSC403 gene of the invention. With the probe thus constructed,expression of the genes of the invention in various neoplastic and othertissues can be detected.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph substituting for a drawing which shows adistribution of the TSC403 gene of the invention in human tissues asfound by the Northern blot analysis in accordance with Example 1-(2).

FIG. 2 is a photograph substituting for a drawing which shows theresults of the RT-PCR analysis of various normal tissues and cancertissues in accordance with Example 1-(4).

FIG. 3 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example 1-(5).

FIG. 4 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example 1-(5).

FIG. 5 is a photograph substituting for a drawing which shows theexpression of the TSC403 gene in human tissues as found by the Northernanalysis in accordance with Example

FIG. 6 is a photograph substituting for a drawing which shows controlcells in the focus forming test in accordance with Example 1-(6).

FIG. 7 is a photograph substituting for a drawing which showstransformant cells as transformed with the TSC403 gene of the inventionin the focus forming test in accordance with Example 1-(6).

FIG. 8 shows the result of a homology study between the predicted aminoacid sequence of the protein encoded by the human ING1L gene of theinvention and that of p33^(ING1) [GenBank A. C. No. AF044076].

FIG. 9 is a photograph substituting for a drawing which shows theresults of a Northern blot analysis of 16 human adult organ-derivedcells in accordance with Example 2-(2).

FIG. 10 is a photograph substituting for a drawing which shows theresults of a Northern blot analysis of the colorectal cancer patienttissue in accordance with Example 2-(2).

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are intended to illustrate the invention infurther detail.

EXAMPLE 1 TSC403 gene

(1-1) Procedure for Imaging by [γ-³³P]ATP Labeling

For confirmation of the human gene expressed by a tissue-specifictechnique, the [-³³P]ATP-labeled imaging method was used. This methodwas essentially carried out according to the protocol of Liang [Liang P., et al., Science, 257, 967-971 (1992)].

Thus, the poly A RNA (0.2 μg) isolated from each of 13 human tissues(adult brain, fetal brain, lung, liver, stomach, pancreas, spleen,mammary gland, prostate, placenta, testis, kidney and heart; Clontech)was mixed with 25 pmol of 3′-anchored oligo-dT primer G(T) 15 MA (Mstands for a mixture of G, A and C) in 8 μl of diethylpyrocarbonate-treated water and the mixture was heated at 65° C. for 5minutes. To this solution, 4 μl of 5×first strand buffer (BRL), 2 μl of0.1 M DTT (BRL), 1 μl of 250 mM dNTPs (BRL), 1 μl of ribonucleaseinhibitor (40 units; Toyobo) and 1 μl of Superscript II reversetranscriptase (200 units; BRL) were added. The final volume of eachreaction mixture was 20 μl. Each solution was incubated at 37° C. for 1hour and diluted 2.5-fold by adding 30 μl of distilled water and thedilution was stored at −20° C. until used.

The cDNA was amplified by PCR in the presence of [γ-³³P]ATP-labeled(Pharmacia) 3′-anchored primer. This PCR amplification of cDNA wascarried out as follows.

Thus, 20 μl of each PCR mixture contained 2 μl of RT reaction mixture, 2μl of 10×PCR buffer (Takara), 4 μl of 2.5 mM dNTPs, 0.25 μl of ExTaq DNApolymerase (5 U/ml; Takara), 25 pmol of 3′-anchored oligo-dT primerlabeled with [α-³³P]ATP, and 25 pmol of 5′-primer (No. 20, a 10-merdeoxyoligonucleotide primer having a randomized sequence of the sequenceSEQ ID NO: 9). The PCR was carried out under the following conditions:one cycle of 95° C. for 3 min., 40° C. for 5 min. and 72° C. for 5 min.;40 cycles of 95° C. for 0.5 min., 40° C. for 2 min. and 72° C. for 1min.; and annealing at 72° C. for 5 min.

The PCR sample was extracted with ethanol, resuspended informamide-sequencing dye, and reacted on the 6% acrylamide-7.5 M ureasequencing gel. The gel was dried without fixing and autoradiography wasperformed overnight.

(1-2) Sub-cloning of the Amplified cDNA Fragment

A 3 MM filter paper, on which the dry gel was placed in advance, wasmarked in a radioactive ink and the autoradiogram was set in registrywith the marking. The gel containing the objective cDNA band was cut outtogether with the 3 MM filter paper and stirred in 300 μl of dH₂O for 1hour. After removal of the polyacrylamide gel and filter paper, the cDNAwas recovered by ethanol precipitation in the presence of 1 μl of 10mg/ml glycogen and 0.3 M NaOAc as the carrier and redissolved in 10 μlof dH₂O. For reamplification, 5 μl of this solution was used. The PCRconditions and primers used were the same as those used for the firstPCR. The reamplification product of a suitable size was recovered as thefirst PCR product and this PCR product was cloned in the Hinc II site ofpUC118 vector (Takara). The nucleic acid sequence was determined byusing ABI377 Automatic Sequencer (Applied BioSystems).

By comparing the various image patterns obtained with the mRNAs isolatedfrom 13 kinds of human tissues, one PCR product expressed specificallyin the lung could be identified. This product was named TSC403DD.

This product consisted of 252 nucleotides. Comparison of this nucleotidedata with the DNA sequences in the GenBank/EMBL Database using FASTAProgram [Person, W. R., et al., Proc. Natl. Acad. Sci., USA, 85,2444-2448 (1988)] revealed that this PCR product has no homology to anyknown DNA sequence.

(1-3) cDNA Screening

A human normal lung cDNA library was constructed using oligo(dT)+randomhexamer-primed human normal lung cDNA and Uni-ZAP™ XR (Stratagene). Allthe 1×10⁶ clones were isolated by the above-described procedure and ascreening was carried out using the [α-³²P]-dCTP-labeled cDNA fragment.Positive clones were selected and the cloned sites of cDNA were excisedin vivo in pBluescript II SK(−).

As a result, about 100 plaques were identified for said TSC403DD. Basedon this result, the transcription amount in the total RNA population wascalculated to be about 0.01%.

The assembled cDNA sequence (TSC403) homologous to TSC403DD comprised3198 nucleotides inclusive of a 1248-nucleotide open reading framecoding for a 416-amino acid residue protein having a calculatedmolecular mass of 44316 Da.

From the primary sequence, this gene product (TSC403 protein) was foundto be a protein containing a transmembrane domain.

Its chromosomal locus was found to be 3q27 where chromosomal aberrationsare noted in various kinds of cancers.

Furthermore, the gene TSC403 of the invention has about 30% homology tohuman lamp-1 and lamp-2.

(2) Expression in Tissues

To delineate the expression profile of TSC403 in tissues, Northernblotting was performed using various human tissues.

In the Northern blot analysis, human MTN (Multiple-Tissue Northern) blotI and II (Clontech) were used. The cDNA fragment was prepared by using aprimer set of T3 and T7 promoter sequences and labeled with [α-³²P]-dCTPby PCR. The membrane containing the amplification product was subjectedto the prehybridization (under the conditions of the product protocol)and further to hybridization according to the product protocol.

After hybridization, the membrane was washed and exposed forautoradiography at −80° C. for 24 hours. The results are shown in FIG.1.

The human tissues used and represented on the drawing are heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon and peripheralblood leukocyte; P.B.L.).

As can be seen from the drawing, a transcript homologous to TSC403 wasspecifically detected in the lung.

(3) FISH

FISH for chromosome mapping was carried out by the known method[Takahashi E., et al., Hum. Genet., 86, 14-16 (1990)] using 0.5 μg ofeach cosmid DNA as a probe. FISH was caught by Provia 100 film (Fuji;ISO 100) or with a CCD camera system (Applied Imaging, Cyto Vision).

As a result, the 100 signals obtained with the cells in the typicalR-band (pro) metaphase were localized in the 3q27 band. Therefore, thechromosomal locus of TSC403 could be identified to be 3q27.

(4) Expression in Cancer Cell Lines and Cancer Tissues as Analyzed byRT-PCR

To investigate whether the expression of the TSC403 gene would bemutated in human cancer cell lines and cancer tissues, four cell lines[Aspc1 (metastatic adenocarcinoma, J. Natl. Cancer Inst., 67, 563-569(1981)), Bxpc3 (adenocarcinoma, undifferentiated; Cancer Invest., 4,15-23 (1986)), MiaPaca2 (adenocarcinoma, Int. J. Cancer, 19, 128-135(1977)) and PANC1 (epithelioid, carcinoma of pancreatic duct, Int. J.Cancer, 15, 741-747 (1975)) and 9 cancer tissues (donated by Dr.Nakamura, The Institute of Medical Science, the University of Tokyo) ]were subjected to RT-PCR analysis.

Thus, 10 μl of the whole RNA isolated from each cell line or cancertissue by using ISOGEN (Wako Chemical Ind.) was treated with 10 units ofRNase-free DNase I (Boehringer Mannheim) for 15 minutes, extracted twicewith phenol-chloroform, and precipitated from ethanol. Thesingle-stranded cDNA was synthesized by means of Superscript I_(TM)RNase H-reverse transcriptase (Life Technology) using oligo-(dT) andrandom primers. A 2 μl-portion of each product was used for PCRamplification.

The primers P1 and P2 having the nucleotide sequences depicted in SEQ IDNO:10 and SEQ ID NO:11 were used for 25-cycle PCR amplification.

The PCR was carried out in 20 μl of a solution of DNA 25 ng, primers 10μM each, dNTP 2.5 mM and Extaq DNA polymerase (Takara) 0.25 U. The PCRproduct was dissolved in ethidium bromide-stained 1.5% agarose gel.

The results of the RT-PCR analysis of 4 cell lines (lane 1=AsPc-1, Lane2=BxPc-3, Lane 3=MIApaca, lane 4=PANC-1), normal pancreatic tissue(Normal pancreas, lanes land 2), pancreatic cancer tissue (Pancreaticcancer, lanes 1-11) and normal lung tissue (Normal lung) by the aboveprocedure are shown in FIG. 2.

It is clear from FIG. 2 that the expression of TSC403 was not found inthe normal pancreatic tissue (Normal pancreas, lanes 1 and 2) but foundexclusively in the pancreatic cancer tissue (Pancreatic cancer, lanes1-11 and Cell lines, lanes 1-4).

Incidentally, the 4 cell lines used above have been deposited with ATCCand their accession numbers are as follows.

Aspc-1; CRL-1682

BxPc-3; CRL-1687

MIApaca; CRL-1420

PANC-1; CRL-1469

(5) Expression of the TSC403 Gene in Various Cancers

(Northern Blot Analysis)

Expression of the TSC403 gene was studied by hybridizing the TSC403 geneto the blot (Invitrogen carrying the following various cancer tissue andnormal tissue mRNA samples (tumor Northern blot analysis). All thecancer tissues and normal tissues used were purchased from Invitrogen.

The results are shown in FIG. 3, FIG. 4 and FIG. 5. The cancer tissuesand normal tissues represented on each drawing are as follows.

FIG. 3:

Brain tumor, brain normal, kidney tumor, kidney normal, liver tumor,liver normal, lung tumor, lung normal, breast tumor, normal breast,uterine tumor, normal uterine, fallopian tube tumor, normal fallopiantube, ovarian tumor, normal ovary.

FIG. 4:

Esophagus tumor, normal esophagus, stomach tumor, normal stomach, colontumor, normal colon, rectum tumor, normal rectum, thyroid tumor, normalthyroid, adrenal tumor, normal adrenal, parotid tumor, normal parotid,lymphoma, normal lymph node.

FIG. 5:

Kidney tumor, normal kidney, ureter tumor, normal ureter, bladder tumor,normal bladder, stomach tumor, normal stomach, ovarian tumor (4 cases),ovarian normal (4 cases).

From the above drawings, significant expression of TSC403 can be foundin the normal lung. In addition, expression of the TSC403 gene wasobserved in breast tumor (FIG. 3), fallopian tube tumor (FIG. 3),esophagus tumor (FIG. 4), colon tumor (FIG. 4), rectum tumor (FIG. 4),thyroid tumor (FIG. 4), parotid tumor (FIG. 4), ureter tumor (FIG. 5),ovarian tumor (FIG. 5, 2 out of 4 cases).

(6) Focus forming Assay by Expression of the TSC403 Gene

The full-length open reading frame of the TSC403 gene was ligated to theBamHI-XhoI site of the pCDNA 3.1/His (Invitrogen) vector to obtain aTSC403 gene expression vector.

Then, using the expression vector obtained above, a focus forming assayusing NIH3T3 cells was carried out according to the method described inthe literature [Shin, C., Shilo, B., Goldfarb, M. P., et al., Proc.Natl. Acad. Sci., USA., 76, 5714-5718 (1979)] to see whether the TSC403gene has a tumorigenic effect on cells.

The results are shown in FIG. 6 (control cells not transformed withTSC403 gene) and FIG. 7 (cells transformed with TSC403 gene).

As is apparent from comparison of the two figures, a definite focus wasformed, as shown in FIG. 7, when the TSC403 gene was force-expressed incells by introduction of the gene. It is, thus, apparent that the TSC403gene, when over-expressed by force, causes a loss of sensitivity to thecontact inhibition phenomenon which is one of malignant transformationsof cells, thus being deeply involved in the tumorigenesis of cells.

EXAMPLE 2 Human ING1L gene

(1) Cloning and DNA Sequencing of the Human ING1L Gene

By the sequencing of cDNA clones arbitrarily selected from a human fetalbrain cDNA library and database search, one clone (GEN-146F11) harboringa cDNA coding for an amino acid sequence having high homology top33^(ING1), a protein considered to be a tumor-suppressive protein, wasisolated by the following procedure.

Thus, the mRNA extracted from human fetal brain was purchased fromClontech and used as the starting material. From the mRNA, the cDNA wassynthesized and cloned into Vector λZAPII (Stratagene) to construct acDNA library (Otsuka GEN Research Institute, Otsuka Pharmaceutical). Bythe in vivo excision method [Short, J. M. et al., Nucleic Acids Res.,16, 7583-7600 (1988)], colonies of Escherichia coli harboring the humangene were caused to form on agar medium and picked up at random and E.coli clones harboring the human gene were registered on a 96 -wellmicrotiter plate. The registered clones were stored at −80° C.

Then, each registered clone was cultured in 1.5 ml of LB medium for 24hours, and using a plasmid automatic extractor PI-100 (Kurabo), the DNAwas extracted and purified. The contaminated E. coli RNA was decomposedby RNase treatment and removed. Finally, the DNA was dissolved to 30 μland a 2 μl portion was used for a rough estimation of DNA size andamount by the minigel method. Another 7 portion was used for sequencingreaction and the remaining 21 μl portion was stored as plasmid DNA at 4°C.

Then, a Sanger's dideoxy terminator [Sanger, F., et al., Proc. Natl.Acad. Sci., U.S.A., 74, 5463-5467 (1977)] using T3, T7 or a syntheticoligonucleotide primer or a cycle sequencing method which is the dideoxyterminator method combined with the PCR method [Carothers, A. M., etal., Bio. Techniques, 7, 494-499 (1989)] was carried out. These are themethods for chain extension reaction with termination specific to 4kinds of bases using a small amount of plasmid DNA (ca 0.1-0.5 g) as atemplate.

Using an FITC (fluorescein isothiocyanate)-labeled primer as thesequence primer, about 25 cycles of reaction using Taq polymerase werecarried out. Of the fluorescence-labeled DNA fragment, the sequence ofabout 400 nucleotides from the 5′-end of the cDNA was determined withthe automatic DNA sequencer ALF™ DNA Sequencer (Pharmacia).

The 3′-nontranslated region is high in heterogeneity among genes andsuited for differentiation of individual genes. Therefore, sequencing ofthe 3′-end region was also performed in some cases.

The huge nucleotide sequence information generated with the DNAsequencer was transmitted to the 64-bit computer DEC3400 forcomputerized homology analysis. This homology analysis was carried outby a database (GenBank, EMBL) search according to UWGCG's FASTA Program[Pearson, W. R. and Lipman, D. J., Proc. Natl. Acad. Sci., USA., 85,2444-2448 (1988)].

The above method of analysis for a human fetal brain cDNA library isdescribed in detail by Fujiwara et al. [Fujiwara, T., et al., DNA Res.,2, 107-111 (1991)].

About 5040 ESTs (expressed sequence tags: partial DNA sequences ofexpressed gene fragment) randomly selected from the human fetal braincDNA library constructed as above were then sequenced.

The clone named GEN-146F11 in the GenBank/EMBL sequence search accordingto the FASTA Program was found to harbor a gene coding for an amino acidsequence having high homology to p33^(ING1) [GenBank A. C. No.AF001954].

To clarify the full-length sequence in said GEN-146F11 clone, a DNAsequencing reaction using T7DNA polymerase and a synthetic primer wascarried out. In addition, using a double-stranded DNA inserted into avector (pBluescript vector; Stratagene) as a template and a syntheticoligonucleotide as a primer, the nucleotide sequence of the cDNAinclusive of the whole coding region was determined by Sanger's dideoxychain termination method, and the sequence was compared with the DNAsequences of several other related genes.

SEQ ID NO:7 shows the nucleic acid sequence of GEN-146F11 clone (cDNA);SEQ ID NO:6 shows the nucleic acid sequence of the coding region of saidclone; and SEQ ID NO:5 shows the deduced amino acid sequence encoded bysaid nucleic acid sequence.

In the above nucleotide sequences, the initiation signal sequence wasfound in the position 92-94and suspected to be the translation startcodon. The predicted stop codon was found in the position 932-934.

The cDNA has a length of 1078 nucleotides and contained an open readingframe of 840 base pairs that coded for a predicted 280-amino acidresidue protein.

By the homology search using FASTA Program, this gene was found to codean amino acid sequence having high homology to p33^(ING1) [GenBank A. C.No. AF044076]. The homology of the nucleotide sequence was 60.0%.

On the amino acid sequence level, the homology between the deduced aminoacid sequence of the protein encoded by the gene of the invention andthe sequence of p33^(ING1) [GenBank A. C. No. AF044076] wasinvestigated. The result is shown in FIG. 8.

FIG. 8 shows the amino acid sequence depicted in single letters; the toprow represents the sequence of the human ING1L protein encoded by thegene of the invention (indicated as hING1L) and the bottom rowrepresents the sequence of p33^(ING1) [Garkavetsev, et al., Nature.Genet., 14, 415-420 (1996); Garkavetsev, etal., Mol. Cell. Biol., 17,2014-2019 (1997), GenBank A. C. No. AF001954; however this sequence hasbeen revised subsequently and the sequence as corrected is shown inGenBank A. C. No. AF044076; indicated as p33^(ING1) on the drawing].

Furthermore, on the same drawing, the solid area (black frame) indicatesthe identical amino acid residues and the shaded area (shaded frame)indicates analogous amino acid residues. The symbol - - - in the hING1Lrow stands for a gap.

It can be seen from the drawing that the amino acid sequence encoded bythe gene of the invention has 58.9% (as calculated based on the sequenceas corrected of p33^(ING1)) homology to the amino acid sequence ofp33^(ING1).

(2) Northern Blot Analysis

The expression of human ING1L mRNA in normal human tissues was evaluatedby Northern blotting using a human cDNA clone labeled by the randomoligonucleotide priming method as a probe.

The Northern blot analysis was performed using a human MTN blot (HumanMultiple Tissue Northern Blot; Clontech) according to the productprotocol.

Thus, the full-length sequence of said clone GEN-146F11 wasPCR-amplified and the PCR product was labeled with [³²P]-dCTP (RandomPrimed DNA Labeling Kit, Boehringer Mannheim) for use as a probe.

The blot was subjected to 4-hour prehybridization and, then, tohybridization in a solution of 50% formamide/5×SSC/10×Decherdsolution/2% SDS solution (containing 100 μg/ml of denatured salmon spermDNA) at 42° C. overnight. After two washings with 2×SSC/0.1% SDS at roomtemperature, 2 washings were carried out with 0.2×SSC/0.1% SDS at 65° C.for 15 minutes. The filter was exposed against X-ray film (Kodak) at−70° C.

The results of 18-hour exposure are shown in FIG. 9.

As can be seen in FIG. 9, the expression was found in all the 16 humanadult organ-derived tissues tested (heart, brain, placenta, lung, liver,skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis,uterus, small intestine, colon, peripheral blood and leukocyte; the samenomenclature applies to the legends on the drawing), and two transcriptsof 1.5 kb and 1.3 kb were detected.

Furthermore, for the several tumor tissues of colorectal cancer, cancerof esophagus, cancer of uterine tube and stomach cancer, too, a similarNorthern blot analysis was carried out using a human TP blot (HumanTumor Panel Blot; Invitrogen) in accordance with the product protocol.

The results in colorectal cancer patient tissues are shown in FIG. 10.

On the drawing, T represents the colorectal tumor tissue (indicated asT:Tumor on the drawing) and N represents the normal colorectal tissue(indicated as N:Normal on the drawing). One set of T and N is the tissuederived from one patient and the drawing shows the results for tissuesderived from 4 patients.

It is clear from the drawing that in each individual patient, the levelof expression of the human ING1L gene is elevated in the canceroustissue as compared with the normal tissue.

Based on the above findings, it is thought that the human ING1L gene ofthe invention is useful for cancer research and therapy, particularlyfor application to cancer diagnosis, and that if any antagonisticinhibitor of expression products of the human ING1L gene be developed inthe future, it should find application as an anticancer agent.

(3) Chromosome Mapping by FISH and Radiation Hybridizing Techniques

FISH for chromosome alignment was carried out by the known procedure[Takahashi, E. et al., Hum. Genet., 86, 14-16 (1990)] using 0.5 μg ofeach cosmid DNA as a probe. FISH was caught by Provia 100 film (Fuji,ISO 100) or with a CCD Camera System (Applied Imaging, Cyto Vision).

As a result, signals of 100 typical R-band (pro) metaphase cellsindicated that the locus of the human ING1L gene on a chromosome was4q35.1.

INDUSTRIAL APPLICABILITY

In accordance with the invention, there is provided not only a novellung-specific gene TSC403 but also a protein encoded thereby. Throughutilization thereof, there is provided a technology by which more lightmay be cast on cancers, e.g. lung cancer and pancreatic cancer, and theprocess of oncogenesis and which finds application in the diagnosis,prophylaxis and therapy thereof.

Further provided in accordance with the invention is a novel human ING1Lgene which enables detection of the expression of the gene in varioustissues, production of a human ING1L protein, which is the expressionproduct of the gene, by genetic engineering technology, and constructionof a specific antibody against said protein. These, in turn, enableresearch into the cell cycle, inhibition of growth or activation ofvarious cells, study of metabolic aging and apoptosis of cells, andexploration, treatment or diagnosis of related diseases such as cancers,as mentioned hereinbefore. In addition, the invention enables thedevelopment of, or screening for, antagonistic inhibitors of said humanING1L protein, namely cell growth suppressants and anticancer drugs.

What is claimed is:
 1. An isolated DNA molecule comprising nucleotides 1 to 1248 of SEQ ID NO.
 2. 2. The isolated DNA molecule of claim 1, wherein said DNA molecule encodes a TSC403 polypeptide comprising amino acids 1-416 of SEQ ID NO:
 1. 